#include "sam.h"
#include "faidx.h"
#include "bam_maqcns.h"
+#include "bam_mcns.h"
#include "khash.h"
#include "glf.h"
#include "kstring.h"
if (p->is_head) printf("^%c", p->b->core.qual > 93? 126 : p->b->core.qual + 33);
if (!p->is_del) {
int j, rb, c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos)];
- rb = (ref && (int)pos < ref_len)? ref[pos] : 'N';
+ rb = (ref && pos < ref_len)? ref[pos] : 'N';
if (c == '=' || toupper(c) == toupper(rb)) c = bam1_strand(p->b)? ',' : '.';
else c = bam1_strand(p->b)? tolower(c) : toupper(c);
putchar(c);
if (p->indel > 0) {
- putchar('+'); putw(p->indel, stdout);
+ printf("+%d", p->indel);
for (j = 1; j <= p->indel; ++j) {
c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos + j)];
putchar(bam1_strand(p->b)? tolower(c) : toupper(c));
}
} else if (p->indel < 0) {
- putw(p->indel, stdout);
+ printf("%d", p->indel);
for (j = 1; j <= -p->indel; ++j) {
c = (ref && (int)pos+j < ref_len)? ref[pos+j] : 'N';
putchar(bam1_strand(p->b)? tolower(c) : toupper(c));
d->max_depth = 0;
d->tid = -1; d->mask = BAM_DEF_MASK;
d->c = bam_maqcns_init();
+ d->c->is_soap = 1; // change the default model
d->ido = bam_maqindel_opt_init();
- while ((c = getopt(argc, argv, "st:f:cT:N:r:l:d:im:gI:G:vM:S2aR:P")) >= 0) {
+ while ((c = getopt(argc, argv, "st:f:cT:N:r:l:d:im:gI:G:vM:S2aR:PA")) >= 0) {
switch (c) {
case 'a': d->c->is_soap = 1; break;
+ case 'A': d->c->is_soap = 0; break;
case 's': d->format |= BAM_PLF_SIMPLE; break;
case 't': fn_list = strdup(optarg); break;
case 'l': fn_pos = strdup(optarg); break;
case 'f': fn_fa = strdup(optarg); break;
case 'T': d->c->theta = atof(optarg); break;
case 'N': d->c->n_hap = atoi(optarg); break;
- case 'r': d->c->het_rate = atof(optarg); break;
+ case 'r': d->c->het_rate = atof(optarg); d->ido->r_snp = d->c->het_rate; break;
case 'M': d->c->cap_mapQ = atoi(optarg); break;
case 'd': d->max_depth = atoi(optarg); break;
case 'c': d->format |= BAM_PLF_CNS; break;
fprintf(stderr, "Usage: samtools pileup [options] <in.bam>|<in.sam>\n\n");
fprintf(stderr, "Option: -s simple (yet incomplete) pileup format\n");
fprintf(stderr, " -S the input is in SAM\n");
- fprintf(stderr, " -a use the SOAPsnp model for SNP calling\n");
+ fprintf(stderr, " -A use the MAQ model for SNP calling\n");
fprintf(stderr, " -2 output the 2nd best call and quality\n");
fprintf(stderr, " -i only show lines/consensus with indels\n");
fprintf(stderr, " -m INT filtering reads with bits in INT [%d]\n", d->mask);
fprintf(stderr, " -t FILE list of reference sequences (force -S)\n");
fprintf(stderr, " -l FILE list of sites at which pileup is output\n");
fprintf(stderr, " -f FILE reference sequence in the FASTA format\n\n");
- fprintf(stderr, " -c output the maq consensus sequence\n");
+ fprintf(stderr, " -c output the SOAPsnp consensus sequence\n");
fprintf(stderr, " -v print variants only (for -c)\n");
fprintf(stderr, " -g output in the GLFv3 format (suppressing -c/-i/-s)\n");
fprintf(stderr, " -T FLOAT theta in maq consensus calling model (for -c/-g) [%f]\n", d->c->theta);
* mpileup *
***********/
-static int mpileup(int n, char **fn)
+#define MPLP_VCF 0x1
+#define MPLP_VAR 0x2
+#define MPLP_AFS 0x4
+#define MPLP_AFALL 0x8
+
+#define MPLP_AFS_BLOCK 0x10000
+
+typedef struct {
+ int max_mq, min_mq, prior_type, flag;
+ double theta;
+ char *reg, *fn_pos;
+ faidx_t *fai;
+ kh_64_t *hash;
+} mplp_conf_t;
+
+typedef struct {
+ bamFile fp;
+ bam_iter_t iter;
+ int min_mq;
+} mplp_aux_t;
+
+static int mplp_func(void *data, bam1_t *b)
+{
+ mplp_aux_t *ma = (mplp_aux_t*)data;
+ int ret;
+ do {
+ ret = ma->iter? bam_iter_read(ma->fp, ma->iter, b) : bam_read1(ma->fp, b);
+ } while (b->core.qual < ma->min_mq && ret >= 0);
+ return ret;
+}
+
+static int mpileup(mplp_conf_t *conf, int n, char **fn)
{
- bamFile *fp;
- int i, tid, pos, *n_plp;
+ mplp_aux_t **data;
+ mc_aux_t *ma = 0;
+ int i, tid, pos, *n_plp, beg0 = 0, end0 = 1u<<29, ref_len, ref_tid;
const bam_pileup1_t **plp;
bam_mplp_t iter;
bam_header_t *h = 0;
- fp = calloc(n, sizeof(void*));
+ uint64_t N = 0;
+ char *ref;
+ khash_t(64) *hash = 0;
+ // allocate
+ data = calloc(n, sizeof(void*));
plp = calloc(n, sizeof(void*));
n_plp = calloc(n, sizeof(int*));
+ // read the header and initialize data
for (i = 0; i < n; ++i) {
bam_header_t *h_tmp;
- fp[i] = bam_open(fn[i], "r");
- h_tmp = bam_header_read(fp[i]);
+ data[i] = calloc(1, sizeof(mplp_aux_t));
+ data[i]->min_mq = conf->min_mq;
+ data[i]->fp = bam_open(fn[i], "r");
+ h_tmp = bam_header_read(data[i]->fp);
+ if (conf->reg) {
+ int beg, end;
+ bam_index_t *idx;
+ idx = bam_index_load(fn[i]);
+ if (idx == 0) {
+ fprintf(stderr, "[%s] fail to load index for %d-th input.\n", __func__, i+1);
+ exit(1);
+ }
+ if (bam_parse_region(h_tmp, conf->reg, &tid, &beg, &end) < 0) {
+ fprintf(stderr, "[%s] malformatted region or wrong seqname for %d-th input.\n", __func__, i+1);
+ exit(1);
+ }
+ if (i == 0) beg0 = beg, end0 = end;
+ data[i]->iter = bam_iter_query(idx, tid, beg, end);
+ bam_index_destroy(idx);
+ }
if (i == 0) h = h_tmp;
else {
// FIXME: to check consistency
bam_header_destroy(h_tmp);
}
}
- iter = bam_mplp_init(n, bam_read1, fp);
- while (bam_mplp_auto(iter, &tid, &pos, n_plp, plp) > 0) {
- printf("%s\t%d\tN", h->target_name[tid], pos + 1);
+ if (conf->fn_pos) hash = load_pos(conf->fn_pos, h);
+ // write the VCF header
+ if (conf->flag & MPLP_VCF) {
+ kstring_t s;
+ s.l = s.m = 0; s.s = 0;
+ puts("##fileformat=VCFv4.0");
+ puts("##INFO=<ID=DP,Number=1,Type=Integer,Description=\"Total read depth\">");
+ puts("##INFO=<ID=AF,Number=1,Type=Float,Description=\"Non-reference allele frequency \\argmax_f P(D|f)\">");
+ puts("##INFO=<ID=AFE,Number=1,Type=Float,Description=\"Expected non-reference allele frequency\">");
+ puts("##FILTER=<ID=Q13,Description=\"All min{baseQ,mapQ} below 13\">");
+ kputs("#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\tFORMAT", &s);
for (i = 0; i < n; ++i) {
- int j;
- printf("\t%d\t", n_plp[i]);
- if (n_plp[i] == 0) printf("*\t*");
- else {
- for (j = 0; j < n_plp[i]; ++j)
- pileup_seq(plp[i] + j, pos, 0, 0);
- putchar('\t');
+ const char *p;
+ kputc('\t', &s);
+ if ((p = strstr(fn[i], ".bam")) != 0)
+ kputsn(fn[i], p - fn[i], &s);
+ else kputs(fn[i], &s);
+ }
+ puts(s.s);
+ free(s.s);
+ }
+ // mpileup
+ if (conf->flag & MPLP_VCF) {
+ ma = mc_init(n);
+ mc_init_prior(ma, conf->prior_type, conf->theta);
+ }
+ ref_tid = -1; ref = 0;
+ iter = bam_mplp_init(n, mplp_func, (void**)data);
+ while (bam_mplp_auto(iter, &tid, &pos, n_plp, plp) > 0) {
+ if (conf->reg && (pos < beg0 || pos >= end0)) continue; // out of the region requested
+ if (hash) {
+ khint_t k;
+ k = kh_get(64, hash, (uint64_t)tid<<32 | pos);
+ if (k == kh_end(hash)) continue;
+ }
+ if (tid != ref_tid) {
+ free(ref);
+ if (conf->fai) ref = fai_fetch(conf->fai, h->target_name[tid], &ref_len);
+ ref_tid = tid;
+ }
+ if (conf->flag & MPLP_VCF) {
+ mc_rst_t r;
+ int j, _ref0, depth, rms_q, _ref0b, is_var = 0, qref = 0, level = 2, tot;
+ uint64_t sqr_sum;
+ if (conf->flag & MPLP_AFS) level = 3;
+ _ref0 = _ref0b = (ref && pos < ref_len)? ref[pos] : 'N';
+ _ref0 = bam_nt16_nt4_table[bam_nt16_table[_ref0]];
+ tot = mc_cal(_ref0, n_plp, plp, ma, &r, level);
+ if (tot) { // has good bases
+ double q;
+ is_var = (r.p_ref < .5);
+ q = is_var? r.p_ref : 1. - r.p_ref;
+ if (q < 1e-308) q = 1e-308;
+ qref = (int)(-3.434 * log(q) + .499);
+ if (qref > 99) qref = 99;
+ }
+ if ((conf->flag & MPLP_VAR) && !is_var) continue;
+ ++N; // number of processed lines
+ printf("%s\t%d\t.\t%c\t", h->target_name[tid], pos + 1, _ref0b);
+ if (is_var) {
+ if (_ref0 == r.ref) putchar("ACGTN"[r.alt]);
+ else printf("%c,%c", "ACGTN"[r.ref], "ACGTN"[r.alt]);
+ } else putchar('.');
+ printf("\t%d\t", qref);
+ if (!tot) printf("Q13\t");
+ else printf(".\t");
+ for (i = depth = 0, sqr_sum = 0; i < n; ++i) {
+ depth += n_plp[i];
for (j = 0; j < n_plp[i]; ++j) {
- const bam_pileup1_t *p = plp[i] + j;
- int c = bam1_qual(p->b)[p->qpos] + 33;
- if (c > 126) c = 126;
- putchar(c);
+ int q = plp[i][j].b->core.qual;
+ if (q > conf->max_mq) q = conf->max_mq;
+ sqr_sum += q * q;
+ }
+ }
+ rms_q = (int)(sqrt((double)sqr_sum / depth) + .499);
+ printf("DP=%d;MQ=%d", depth, rms_q);
+ if (tot) {
+ printf(";AF=%.3lf", 1. - r.f_em);
+ if (level >= 2) printf(";AFE=%.3lf", 1-r.f_exp);
+ if (conf->flag & MPLP_AFALL) {
+ printf(";AF0=%.3lf;AFN=%.3lf", 1-r.f_naive, 1-r.f_nielsen);
+ if (conf->flag & MPLP_AFS) printf(";AFB=%.3lf", 1-r.f_map);
}
}
+ printf("\tGT:GQ:DP");
+ if (tot) {
+ for (i = 0; i < n; ++i) {
+ int x = mc_call_gt(ma, r.f_exp, i);
+ printf("\t%c/%c:%d:%d", "10"[((x&3)==2)], "10"[((x&3)>0)], x>>2, n_plp[i]);
+ }
+ } else for (i = 0; i < n; ++i) printf("\t./.:0:0");
+ putchar('\n');
+ if (N % MPLP_AFS_BLOCK == 0) mc_dump_afs(ma);
+ } else {
+ printf("%s\t%d\t%c", h->target_name[tid], pos + 1, (ref && pos < ref_len)? ref[pos] : 'N');
+ for (i = 0; i < n; ++i) {
+ int j;
+ printf("\t%d\t", n_plp[i]);
+ if (n_plp[i] == 0) printf("*\t*");
+ else {
+ for (j = 0; j < n_plp[i]; ++j)
+ pileup_seq(plp[i] + j, pos, ref_len, ref);
+ putchar('\t');
+ for (j = 0; j < n_plp[i]; ++j) {
+ const bam_pileup1_t *p = plp[i] + j;
+ int c = bam1_qual(p->b)[p->qpos] + 33;
+ if (c > 126) c = 126;
+ putchar(c);
+ }
+ }
+ }
+ putchar('\n');
}
- putchar('\n');
}
+ if ((conf->flag&MPLP_VCF) && (conf->flag&MPLP_AFS)) mc_dump_afs(ma);
+ if (hash) { // free the hash table
+ khint_t k;
+ for (k = kh_begin(hash); k < kh_end(hash); ++k)
+ if (kh_exist(hash, k)) free(kh_val(hash, k));
+ kh_destroy(64, hash);
+ }
+ mc_destroy(ma);
bam_mplp_destroy(iter);
bam_header_destroy(h);
- for (i = 0; i < n; ++i) bam_close(fp[i]);
- free(fp); free(plp);
+ for (i = 0; i < n; ++i) {
+ bam_close(data[i]->fp);
+ if (data[i]->iter) bam_iter_destroy(data[i]->iter);
+ free(data[i]);
+ }
+ free(data); free(plp); free(ref); free(n_plp);
return 0;
}
int bam_mpileup(int argc, char *argv[])
{
+ int c;
+ mplp_conf_t mplp;
+ memset(&mplp, 0, sizeof(mplp_conf_t));
+ mplp.max_mq = 60;
+ mplp.prior_type = MC_PTYPE_FULL;
+ mplp.theta = 1e-3;
+ while ((c = getopt(argc, argv, "vVcF2Sf:r:l:VM:q:t:")) >= 0) {
+ switch (c) {
+ case 't': mplp.theta = atof(optarg); break;
+ case '2': mplp.prior_type = MC_PTYPE_COND2; break;
+ case 'f':
+ mplp.fai = fai_load(optarg);
+ if (mplp.fai == 0) return 1;
+ break;
+ case 'r': mplp.reg = strdup(optarg); break;
+ case 'l': mplp.fn_pos = strdup(optarg); break;
+ case 'V':
+ case 'c': mplp.flag |= MPLP_VCF; break;
+ case 'S': mplp.flag |= MPLP_AFS; break;
+ case 'F': mplp.flag |= MPLP_AFALL; break;
+ case 'v': mplp.flag |= MPLP_VAR; break;
+ case 'M': mplp.max_mq = atoi(optarg); break;
+ case 'q': mplp.min_mq = atoi(optarg); break;
+ }
+ }
if (argc == 1) {
- fprintf(stderr, "Usage: samtools mpileup <in1.bam> [<in2.bam> [...]]\n");
+ fprintf(stderr, "\n");
+ fprintf(stderr, "Usage: samtools mpileup [options] in1.bam [in2.bam [...]]\n\n");
+ fprintf(stderr, "Options: -f FILE reference sequence file [null]\n");
+ fprintf(stderr, " -r STR region in which pileup is generated [null]\n");
+ fprintf(stderr, " -l FILE list of positions (format: chr pos) [null]\n");
+ fprintf(stderr, " -M INT cap mapping quality at INT [%d]\n", mplp.max_mq);
+ fprintf(stderr, " -q INT filter out alignment with MQ smaller than INT [%d]\n", mplp.min_mq);
+ fprintf(stderr, " -t FLOAT scaled mutation rate [%lg]\n", mplp.theta);
+ fprintf(stderr, " -c generate VCF output (consensus calling)\n");
+ fprintf(stderr, " -v show variant sites only\n");
+ fprintf(stderr, " -S calculate AFS (slow, to stderr)\n");
+ fprintf(stderr, " -2 conditional prior\n");
+ fprintf(stderr, "\n");
+ fprintf(stderr, "Notes: Assuming error independency and diploid individuals.\n\n");
return 1;
}
- mpileup(argc - 1, argv + 1);
+ mpileup(&mplp, argc - optind, argv + optind);
+ free(mplp.reg);
+ if (mplp.fai) fai_destroy(mplp.fai);
return 0;
}